﻿Three new taxa of the lichen genus Lobothallia (Megasporaceae, Ascomycota) from China

﻿Abstract Two new species; Lobothalliacrenulata Lun Wang & Y. Y. Zhang, L.lobulata Lun Wang & Y. Y. Zhang and one new variety; L.subdiffractavar.rimosa Lun Wang & Y. Y. Zhang, are reported from China and described, based on morphological, chemical and molecular characters. Phylogenetic analyses showed that these new taxa form monophyletic groups. Lobothalliacrenulata and L.lobulata, together with L.hydrocharis, L.radiosa and L.recedens, form a well-supported clade, whereas L.subdiffractavar.rimosa is nested within the samples of L.subdiffracta. Lobothalliacrenulata is characterised by its placodioid thallus, thickly pruinose upper surface with a rimose appearance, aspicilioid to lecanorine apothecia with a crenate thalline margin and concave, black and pruinose discs. Lobothallialobulata is characterised by its placodioid thallus, pruinose upper surface with lobules, aspicilioid when immature, lecanorine to zeorine apothecia at maturity and concave to plane, dark brown, shiny and epruinose discs. Lobothalliasubdiffractavar.rimosa is characterised by its areolate thallus, rimose and pruinose upper surface, lecanorine apothecia and slightly concave to plane, black and pruinose discs. Secondary metabolites were not detected in the two new species nor the new variety. A key is provided for the species of Lobothallia in China.


Morphological and chemical examination
In this study, 108 specimens were examined and deposited in the following Herbaria: Anhui Normal University (AHUB), Lichen Herbarium, Kunming Institute of Botany, Chinese Academy of Sciences (KUN-L) and Shandong Normal University (SDNU).The external morphological characters of air-dried material were studied under a stereomicroscope (OLYMPUS SZ61TR).Anatomical features were studied using a light microscope (OLYMPUS BX43) on transverse sections of apothecia and thalli, prepared manually with a razor blade and mounted in water or lactophenol cotton blue (LCB).Spore measurements were presented as: (minimum-) (x ¯ -SD) -x ¯ -(x ¯ + SD) (-maximum), where x ¯ is the arithmetic mean and SD is the standard deviation (values were rounded to the nearest 0.5 µm), followed by the number of measurements (n) (Li et al. 2023).Lugol's solvent (I) was used to examine the apical structure of asci.Crystals in apothecia and thallus were observed in polarised light (POL) and their solubility was assessed in 10% potassium hydroxide (KOH) (K).Spot tests were conducted using K and a saturated aqueous solution of sodium hypochlorite (NaClO) (C).Secondary metabolites were analysed using thin layer chromatography (TLC) with the solvent C (Orange et al. 2001).

Phylogenetic analyses
The raw sequences were initially checked with the BLAST tool on the NCBI online service (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to confirm the lichen affinity.Geneious v.8.0.was used to assemble and edit the raw sequences and generate a single matrix for nrITS and mtSSU.Each matrix was aligned using the MAFFT v.7 online server (https://mafft.cbrc.jp/alignment/server/).Before concatenating the single-gene matrices of nrITS and mtSSU, we tested for potential incongruity using IQ-TREE with 1000 ultrafast bootstrap replicates.No well supported conflict was detected.SequenceMatrix 1.7.8 (Vaidya et al. 2011) was used to concatenate the nrITS and mtSSU genes and produce a 2-locus dataset.PartitionFinder v.2.0 (Lanfear et al. 2017) was used to estimate the best schemes and nucleotide substitution models for Maximum Likelihood (ML) and Bayesian Inference (BI) analyses.The best-fit models for ITS1, 5.8S, ITS2 and mtSSU were GTR+G.
Phylogenetic relationships were inferred using Bayesian Inference and Maximum Likelihood.The Bayesian method was performed with MrBayes 3.2.7 (Ronquist et al. 2012), using four Markov chains running for 12 million generations.Trees were sampled every 100 generations and the first 25% were discarded as burn-in.Subset rates were modelled as fixed and equal.We used the default distributions for priors.The average standard deviation of split frequencies fell below 0.01 by the end of the analysis.Tracer v.1.7 (Rambaut et al. 2018) was used to assess chain convergence by checking the effective sampling size (ESS > 200).ML analyses were performed with RaxmlGUI (Silvestro and Michalak 2012) using the general time reversible model of nucleotide substitution with the gamma model of rate heterogeneity (GTRGAMMA).All trees were visualised using Mega v.7.0 (Kumar et al. 2016) and edited using PowerPoint.Bayesian posterior probabilities ≥ 0.95 and ML bootstrap values ≥ 70% were presented on the ML tree.

Results and discussion
The nrITS-mtSSU data matrix encompassed a total of 91 sequences (61 nrITS, 30 mtSSU, including 49 downloaded from GenBank and 42 newly generated) from 61 samples of 22 taxa (Table 1).The length of the final aligned dataset was 1342 nucleotides.Three species, Aspicilia cinerea (L.) Körb., Circinaria esculenta (Pall.)Sohrabi and C. fruticulosa (Eversm.)Sohrabi were chosen as the outgroup, following previous phylogenetic studies (Nordin et al. 2010;Paukov et al. 2019).The two-locus phylogenetic tree showed that species of the genus Lobothallia fell into three main clades (Fig. 1).Our two new species, Lobothallia crenulata and L. lobulata, formed highly supported monophyletic clades, which belonged to Clade I. Lobothallia cheresina is the basal species of this clade, differing from other species of this clade by its non-lobate thallus with definite cracks up to the margins and aspicilioid apothecia without promi-nent margin (Müller 1880;Paukov et al. 2019;Zulfiqar et al. 2022).Lobothallia lobulata and L. crenulata, together with the species of L. hydrocharis (Poelt & Nimis) Sohrabi & Nimis, L. radiosa and L. recedens (Taylor) A. Nordin, Savić & Tibell formed a monophyletic subclade.Species of this subclade had no secondary metabolites, with the exceptions of L. radiosa, which has three chemotypes: chemotype parasitica (stictic acid), chemotype subcircinata (norstictic acid) and chemotype radiosa (without norstictic or with trace amount of norstictic acid) (Nimis and Poelt 1987;Ryan 2004;Paukov et al. 2019;Zulfiqar et al. 2022).Lobothallia crenulata is the basal species of this subclade and differs from the other species by its thickly pruinose thallus with rimose upper surface and its crenate thalline margin.Lobothallia lobulata is sister to a subclade formed by L. hydrocharis and L. radiosa, but differs in the presence of lobules at the upper surface and its lecanorine to zeorine apothecia at maturity.
Clade II comprises a single species, Lobothallia elobulata Zulfiqar, Khalid & Paukov, which is easily recognised by its black prothallus, non-lobate thallus with a smooth and epruinose upper surface, lecanorine apothecia with thinly pruinose disc and the absence of secondary metabolites (Zulfiqar et al. 2022).Further research is required regarding the phylogenetic position of this clade.
Clade III consisted of several subclades and species lineages.Species of this clade were mainly distributed in Asia.Our new variety; Lobothallia subdiffracta var.rimosa, together with samples of "L.helanensis" formed a sister group to L. subdiffracta, which was nested within Clade III.These taxa differ from other species of this clade by their non-lobate, thick and areolate thallus, rimose upper surface and absence of secondary metabolites (Magnusson 1944;Kou et al. 2013;Paukov et al. 2019).The species "Lobothallia helanensis" was synonymised to L. subdiffracta by Paukov et al. (2019).Lobothallia subdiffracta var.rimosa differs from L. subdiffracta by its lecanorine apothecia with permanent thalline margin and pruinose discs.
Nine out of the 28 species within the genus Lobothallia have no available gene sequences.Amongst these, secondary metabolites are only absent for Lobothallia chadefaudiana (Cl.Roux) A. Nordin, Cl.Roux & Sohrabi.Lobothallia chadefaudiana can be distinguished from our new taxa by the non-lobate thallus, with rough yellowish granules on the upper surface and immersed apothecia (Roux 1977;Paukov et al. 2019).Lobothallia cernohorskyana Nordin, L. gangwondoana S.Y.Kondr., J.J. Woo & Hur and L. lacteola (Oxner) Şenkard., Paukov, Davydov & Sohrabi differ from the two new species by their non-lobate thallus, aspicilioid apothecia and the presence of norstictic acid (Clauzade and Vězda 1970;Roux et al. 2016;Paukov et al. 2019;Kondratyuk et al. 2020).Lobothallia zogtii is characterised by the brown thallus, white bordered squamules and the presence of stictic acid complex (Paukov et al. 2019).Lobothallia platycarpa shares whitish-grey and lobate thallus with the new species of L. crenulata, but differs in its immersed apothecia and the presence of norstictic acid (Zulfiqar et al. 2022).Lobothallia hedinii could potentially be confused with L. lobulata, but differs by its brown thallus, straight and parallel lobes and presence of norstictic acid (Magnusson 1940;Paukov et al. 2019).Diagnosis.Lobothallia crenulata is characterised by its placodioid, thickly pruinose thallus, rimose upper surface, aspicilioid to lecanorine apothecia with a crenate thalline margin, concave, black and pruinose disc and the absence of secondary metabolites.
Chemistry.K-, C-, KC-.No substances were detected by TLC.Distribution and ecology.This new species grows on calcareous schist rocks at elevations of 3924-4304 m in Xizang Autonomous Region, China.
Notes.The new species is similar to Lobothallia iqbalii Zulfiqar, Khalid & Paukov and L. pakistanica Razzaq, Fayyaz, Khalid & Afshan in its placodioid thallus, white to light grey upper surface and the absence of secondary metabolites.Lobothallia iqbalii differs in its lecanorine apothecia with plane to convex disc and an entire and thick thalline margin (Zulfiqar et al. 2022).Lobothallia pakistanica differs in its rarely cracked central areoles, thinner epinecral layer (8-16 µm), slightly concave to flat, rarely pruinose disc and the absence of thalline margin (Zulfiqar et al. 2022).Lobothallia subdiffracta shares some features with L. crenulata: rimose and pruinose thallus.However, L. subdiffracta differs in its grey thallus with thinner and uneven pruina and its non-lobate thallus (Magnusson 1944;Kou et al. 2013;Paukov et al. 2019).Another taxon, Lobothallia pruinosa, also has a placodioid and pruinose thallus and pruinose discs, but differs from L. crenulata in its entire thalline margin and the presence of norstictic and constictic acids (Kou et al. 2013).
Chemistry.K-, C-, KC-.No substances were detected by TLC.Distribution and ecology.This new species grows on exposed calcareous rocks at elevations of 3262-3296 m elev. in Sichuan Province, China.
Notes.This species shares morphological features with the closely-related Lobothallia radiosa: both have placodioid thallus, areolate in central parts and conspicuously radiate marginal lobes.Lobothallia radiosa has three chemotypes: parasitica with stictic acid, subcircinata with norstictic acid and radiosa with or without a trace amount of norstictic acid (Ryan 2004;Reyim et al. 2012;Paukov et al. 2019).The new species shares the chemotype of some specimens of radiosa, but differs in the presence of lobules, the aspicilioid apothecia when immature, lecanorine to zeorine at maturity and in its phylogenetic position.Lobothallia hydrocharis also has a placodioid thallus with secondary metabolites absent, but differs by its aspicilioid apothecia with black and matt discs and its distribution, which is restricted to Sardinia, Italy (Nimis and Poelt 1987;Nimis 2016;Nascimbene et al. 2023).
Additional specimens examined.

Figure 1 .
Figure 1.Phylogenetic tree generated from Maximum Likelihood (ML) analysis, based on the concatenated nrITS and mtSSU dataset.ML bootstrap values ≥ 70% (left) and Bayesian posterior probabilities ≥ 0.95 (right) are displayed along the branches of the tree.Newly-generated sequences are indicated in bold.The three new taxa are marked by triangles.

Figure 4 .
Figure 4. Lobothallia subdiffracta var.rimosa A thallus and apothecia B thalline margin crenate when immature and entire at maturity C slightly radiate arrangement of marginal areoles D cross-section of thallus E cross-section of thallus under polarised light F vertical section of apothecia G ascus H ascus (Lugol's solution) I ascospores J conidia.All sections were mounted in water except where otherwise stated.Scale bars: 2 mm (A); 1 mm (B, C); 100 µm (D, E); 20 µm (F); 5 µm (G, H, I, J).

Table 1 .
Sequences used in the phylogenetic analyses in this study, with specimen information and GenBank accession numbers.Newly-obtained sequences are in bold font."na" indicates that there is no sequence available.